Gene expression is controlled by cis-regulatory elements (CREs) such as enhancers and promoters that contain binding sites for transcriptional activators and repressors. CREs are stretches of non-coding DNA that control when, where, and at which levels genes are expressed. The overall goal of this proposal is to gain insights into the poorly understood mechanisms that underlie CRE function. Deciphering the rules that underlie their architecture will improve our understanding of the fundamental biological phenomenon of differential gene expression. As mutations in CREs lead to altered gene expression, this proposal is also relevant for gaining insights into the genetic basis of disease. n this proposal, I use the compact Drosophila rhodopsin (rh) promoters as a CRE model system to address the following questions: a) How does the same CRE control gene expression in different tissues at different developmental time points? The same minimal rh promoter regions (less than 300 base pairs) control rh expression in four different developmental and functional contexts: larval photoreceptors, adult photoreceptors, circadian 'eyelet' photoreceptors and auditory neurons. I will take advantage of a large collection of transgenic fly stocks that carry mutant rh promoters, which I have created in the mentor's lab, to decipher the cis-regulatory code in these different contexts (mentored phase). b) What distinguishes CREs that drive gene expression in a subset of a particular cell type from CREs that drive expression in all the cells o the same cell type? I will compare the rh promoters, which control highly restricted expression in subtypes of photoreceptors, to promoters of genes that are expressed in all photoreceptors. I will identify such 'pan-PR' promoters with bioinformatics (mentored phase) and ChIP-seq technology (independent phase). This will allow me to test the hypothesis that these two CRE types share general activator motifs, but rh genes have additional repressor motifs to achieve subtype specificity. The K99 Award will allow me to receive relevant training in ChIP-seq technology that is required for this goal. c) How do CREs achieve robust and uniform levels of gene expression within a particular cell type? Preliminary results suggest that rh genes have distal enhancers that ensure robust and high expression levels. I will identify and dissect the regulatory regions that control quantitative aspects of rh expression (independent phase). This will allow me to compare the 'expression level' code to the 'spatiotemporal' CRE code. d) Do genes with very different functions but common, highly restricted expression patterns use the same cis-regulatory code? Previous studies in the Desplan lab have established that the tumor suppressor warts and the growth regulator melted are re-used in a double-negative feedback loop to mediate an unambiguous decision for expression of either blue-sensitive Rhodopsin 5 (Rh5) or green-sensitive Rhodopsin 6 (Rh6). As melted is expressed in the same photoreceptor subtype as rh5 and warts is co- expressed with rh6, it is an intriguing question whether the same or a different cis-regulatory code is used for subtype-specific expression of melted/rh5 or warts/rh6. I will dissect the warts and melted loci to identify activator and repressor motifs that mediate their specific expression in two different photoreceptor subtypes (independent phase). I will also determine whether the same trans-acting factors are used for subtype-specific expression. This will allow me to compare the CRE code of two independent examples of highly restricted expression in the same cellular subtype. Using the insights gained from the experiments above, I will reconstruct the cis-regulatory logic of the rhodopsin promoters and will test the reconstructed promoters in mutant backgrounds to determine whether they depend on the same transcription factors. Moreover, I will assess whether they drive proper expression in other cellular contexts (see above). Hereby, I will test the completeness of our understanding of the cis- regulatory logic of rhodopsin expression. The training phase of this proposal will be performed in the lab of my mentor Dr. Claude Desplan in the Center for Developmental Genetics at New York University (NYU) in collaboration with the lab of my consultant Dr. Stephen Small. The NYU Center for Developmental Genetics and the nearby Center for Genomics and Systems Biology provide all essential equipment and facilities required for the proposed research. My long- term career goal is to establish an independent research group at an academic institution and to become a leading scientist in the field of gene regulation.